Many membrane proteins, such as G-protein-coupled receptors (GPCRs), play key roles in mediating numerous physiological processes, which makes them important targets for pharmaceutical intervention. While the human genome sequence has revealed the existence of more than eight hundred putative GPCRs to date, the high-resolution structure of only one of these receptors (rhodopsin) has been solved. The inability to derive structural information for more of these proteins stems in part from their low abundance, as well as from difficulties associated with maintaining their activity once they are isolated from their membrane environments. The goal of the proposed research is to improve receptor solubilization and purification protocols and to identify crystallization conditions that do not adversely affect various aspects of receptor activity. To succeed in the the structural analysis of GPCRs, the work will be performed as a collaboration between biochemists, biophysicists, and crystallographers and will focus on receptors involved in HIV biology, the immune response, neural development, and diabetes. The specific aims center on 1) optimizing the isolation of active receptors, 2) identifying conformationally sensitive antibodies for co- crystalization, 3) developing affinity chromatography protocols, 4) evaluating the effects of crystallization conditions on receptor activity, and 5) resolving the kinetics and conformational states of ligand/receptor complexes. Collectively, these aims will lead to the isolation of higher-quality receptors readily amenable to structural analysis and will foster a more systematic approach toward their crystallization. Streamlining crystallography efforts while simultaneously discovering details about receptor function will serve as the basis for developing agents that thwart, control, or cure GPCR-related diseases and conditions.